The long term goal of this project is to prepare novel macrolide antibiotics that are active against methicillin-resistant, macrolide-resistant, Staphylococcus aureus strains, in an attempt to augment chemotherapy of diseases caused by antibiotic-resistant human pathogens. These compounds will simultaneously block protein synthesis by inhibiting the peptidyl transferase (PT) activity of the ribosomes and at the same time overcome the loss of binding of normal macrolides to macrolide-resistant ribosomes by carrying chemical extensions that enable binding to other ribosomal sites. We will test the concept that this approach is feasible by producing a small series of novel molecules that can be chemically modified at different sites to inhibit PT and to bind tightly to ribosomes. The specific aims for the Phase I research are: (1) Construct recombinant microorganisms making a small series of 14-membered macrolides containing the disaccharide mycaminose-mycarose at the C5 position as direct fermentation products. (2) If production of a 14-membered macrolides containing the disaccaride is not possible, construct organisms making a small series of 14-membered macrolides containing the monosaccharide mycaminose at the C5 position. (3) Chemically acylate the sugars of the compounds produced in aims 1 or 2 and determine if PT activity of bacterial ribosomes is inhibited in a variety of assays. (4) Add side chains to the macrolactone backbone of the molecules that inhibit PT activity to enable tight binding to methylated and non-methylated bacterial ribosomes. The lead compounds discovered would be optimized in Phase II research.